Hendrix, J.; Al Mubarak, R.; Rossmassler, K.; Nielsen, H.; Wynn, E.; Moore, C. M.; Jones, I. L.; Voskuil, M. I.; Podell, B. K.; Robertson, G. T.; Wang, C.; Walter, N. D.

Preclinical tuberculosis (TB) drug evaluation relies heavily on mouse infection models, yet drug efficacy varies depending on inoculum and the timing of treatment initiation. These differences reflect, in part, physiological adaptations of Mycobacterium tuberculosis (Mtb) to host immune pressure. We used novel molecular markers of pathogen health (RS ratio and SEARCH-TB) to probe Mtb phenotypes at the pre-treatment baseline of two standard drug evaluation models - the BALB/c high dose aerosol (HDA) and low dose aerosol (LDA) infections - focusing on the transition from innate to adaptive immunity. We found that the onset of adaptive immunity coincided with a rapid reprogramming of Mtb physiology, characterized by suppressed respiration, metabolism, and biosynthesis, together with induction of stress and nutrient acquisition pathways. Changes in key Mtb processes were concordant with changes in host expression of canonical features of adaptive immunity. Our results also explain key model-specific differences in drug efficacy: when used for drug evaluation, the HDA model begins treatment during the innate immune phase when Mtb are metabolically active, whereas the LDA model initiates therapy after host adaptive immunity has already activated and the bacterial population is immune-constrained. The HDA model is shown to be a mixed model, spanning immune phases with initial treatment acting on replicating bacteria and later treatment acting on immune-constrained populations. Together, the two models offer complementary perspectives on therapeutic activity across the spectrum of bacterial states and provide a framework for designing regimens that are effective against both active and immune-constrained Mtb.